High resolution biosensor system

ABSTRACT

A high resolution biosensing system for detecting and identifying a biochemical material to be tested by using proportional relationship between frequency variation of oscillation and mass of the biochemical material to be tested comprises a biosensor: all oscillator for generating oscillation based on the sensed result, a phase-lock loop circuit receiving the oscillation of the oscillator and generating pulse signals; an ultra-high frequency counter for counting the Bulb; signals; and a microprocessor for storing and displaying output from the ultra-high frequency counter and for controlling the oscillator. The phase-lock loop circuit generates the pulse signals of a frequency, which is n times the frequency of the oscillator and with a constant phase difference therebetween to trigger the ultra-high frequency counter. Accordingly, the resolution can be raised up to n times.

BACKGROUND OF TECHNOLOGY

[0001] In the fast developing field of biology and medical technology, it is an obstacle to the advancement that people cannot precisely measure the variation of biological phenomenon in real time to realize the function of the variation. As from 1990, a biosensor system has been defined as an apparatus which utilizes. immobilized biomolecules in combination with a transducer to detect in vivo or in vitro chemicals or produce a response after a specific interaction with the chemicals. The biomolecules comprise molecule identifying elements for the tissue of an organism or an individual cell. Such elements are used for receiving or generating biosensor signals. The transducer is a hardware instrument element that mainly functions as a physical signal converting element. Consequently, a biosensor system can be constituted by combining specific biologically active materials, which can be obtained by isolating, purifying or inventively synthesizing via biochemical methods, with a precise and fast responding physical transducer.

[0002] The earlier biosensor, which was constituted by an enzyme electrode (Clark et al., 1962), such as the enzyme electrode for use in the blood-sugar test, was developed and marketed by the YSI Company. After the year of 1988, the non-shaped and card-shaped enzyme electrodes utilizing a mediator to speed up the time of response, enhance the sensitivity, and reduce the interference caused by other biological materials, have been developed (Demielson et al., 1988). However, the sensitivity of such biosensors, of the first generation, is limited by the weak conjugation between the biomolecule-enzyme and the test target. Even the enzyme possesses an ability of amplifying the signal, there still exists a defect in which a test target of low concentration cannot be detected in a short period of time.

[0003] The second generation biosensor, which is an affinity biosensor, is designed to overcome the above-described obstacles. It adopts anti-body or receptor protein as a molecule identifier. Generally, its conjugation constant between biomolecules and target molecules is above 10⁷ M⁻¹, and its detectable limit value is much more precise and smaller than that of the first generation biosensor.

[0004] The transducer of the second generation biosensor call be made by a field effect transistor (FET), a fiber optic sensors (FOS), a piezoelectric crystal (PZ), a surface acoustic wave (SAW) device, etc. The second generation biosensor was developed by a Swedish company, Pharmacia Biosensor AB, in the year of 1991. They utilize the technologies of micromachining and genetic engineering to develop the affinity biosensors, BIACORE and BIA lite. These products utilize the technologies of surface plasmon resonance (SPR) and micromachining to conduct a real time detection of biomolecules, in general, under the concentration from 10⁻³ g/ml to 10⁻⁹ g/ml to achieve an acceptable resolution. Although these products may achieve high resolution, they are not economical and practical because of their difficult technologies and their high price, for example, US $300,000 dollars. In addition, the price of their consumptive detection chips, which cost $200 US dollars for each chip, is also very expensive. As a result, it is quite difficult to popularize these products,

[0005] Among the second generation biosensors, an alternative one adopts a quartz crystal microbalance (QCM) system of the piezoelectric technology as the transducer. Such an apparatus, which costs about $30,000 US dollars and each consumptive chip of which costs about $30 US dollars, is much cheaper than that of the aforesaid one, which utilizes the technology of SPR. However, its resolution and sensitivity can merely reach 10⁻³ g/ml to 10⁻⁶ g/ml.

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to overcome the defect of QCM, and promote the sensitivity and resolution of the QCM biosensor system, in order to make it more economical and practicable. Furthermore, if the present invention is utilized in combination with a transducer of high precision, the detection resolution can be significantly raised.

[0007] In accordance with the present invention, a high resolution biosensor system applies the factors, such as the density, viscosity and temperature of the gas or liquid present at the surface of a piezoelectric qua:: crystal, as well as the differential pressure between the two sides of the crystal, to influence the oscillation frequency of the crystal. The relation among these factors can be illustrated by the following equation:

ΔF=CF ² ΔM/A+CF ^(⅔)(Δη_(L)Δρ_(L))^(½)

[0008] wherein

[0009] C: a constant, −2.3×10⁻⁶ cm²/Hz-g

[0010] ΔF: the frequency variation caused by mass load

[0011] F: oscillation frequency of quartz crystal

[0012] ΔM: the variation of mass load carried by the electrode

[0013] A: area of electrode

[0014] Δη_(L): variation of solution viscosity

[0015] ρ_(L): variation of solution density

[0016] As the density and viscosity of the solution remain constant, the frequency variation (ΔF) is directly proportional to the variation of the mass load (ΔM). However, the precision of the frequency counter used in traditional biosensor systems can only reach 1 Hz. If the base clock is 10 MHz, the ultimately detectable limit can only be 0.43×10⁻⁹ g (approximately corresponding to 4.3×10⁻⁶ g/ml). The present invention utilizes the phase-lock loop (PLL) circuit to generate a counting signal that has the same phase as the base clocks but the frequency thereof is n times higher than that of the base clock, such that the resolution can be raised n times. For instance, if n=100, the frequency for the resolution can reach 0.01 and the ultimately detectable limit can be up to 4.3×10⁻¹² g (approximately corresponding to 4.3×10⁻⁹ g/ml). This invention may vastly enhance the precision of measurement and improve the identification sensitivity of biological target by up to 100 times, and thus reach the virus level of identification. Furthermore, the PLL circuit comprises a filter for tracing phase error, and utilizes close loop control to maintain the phase relevancy. Therefore, the frequency jittering problem customarily caused by noises in input signals can be overcome, because the output signal of PLL does not disappear with the instantaneous variation, such that the S/N ratio can be raised and a stable output frequency can be achieved.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is schematica diagram of a conventional piezoelectric microbalance biosensor;

[0018]FIG. 2 is schematic of the high resolution biosensing system according to the present invention;

[0019] FIGS. 3 shows an embodiment of practicing the idea shown in FIG. 2;

[0020]FIG. 4 is a block diagram showing the PLL circuit of the present invention;

[0021]FIG. 5 is a wave form diagram for signals at each node of FIG. 4;

[0022]FIG. 6 illustrates the relationship between the input and the output of the PLL circuit according to the present invention.

DESCRIPTION OF THE INVENTION

[0023] One of the embodiments of the present invention is applied in a piezoelectric biosensor which induces an oscillating electric field along a direction perpendicular to the surface of the chip to make The crystal lattice inside the chip produce like mechanic oscillations similar to standing wave. This type of mechanic oscillation can be indicated by a specific frequency. A resonant frequency can be measured by applying an appropriate oscillation circuit.

[0024] The following equation shows the relation ship between the frequency of piezoelectric quartz crystal and the solution of the detected organism:

ΔF=CF ² ΔM/A+CF ^(⅔)(Δη_(L)Δρ_(L))^(½)

[0025] wherein

[0026] C: a constant, −2.3×10⁻⁶ cm²/Hz-g

[0027] ΔF: the frequency variation caused by mass load

[0028] F: oscillation frequency of quartz crystal

[0029] ΔM: the variation of mass load carried by the electrode

[0030] A: area of electrode

[0031] Δη_(L): variation of solution viscosity

[0032] Δρ_(L): variation of solution density

[0033] As the density and viscosity of the solution remain constant, the frequency variation is directly proportional to the mass loads and will be detected by QCM biological detection crystal, then the QCM crystal outputs a signal accordingly. In the prior arts, the customarily known apparatuses transmit said signal to an oscillation circuit to produce an oscillation signal. Subsequently, the oscillation signal of the oscillator is transmitted to a frequency counter to obtain a frequency value. The principle of the counter is to sum up the counts of the input pulses every second to figure out the frequency value (the sum of pulses per second). For example, if the pulses are summed up per second, the minimum unit (resolution) is 1 Hz. If they are summed up per ten seconds, the minimum unit is 0.1 Hz (namely, 0.1 pulse per second). As a result, for the purpose of promoting resolution, the sampling rate must be compromised. If the sampling rate is remained as summing up once per second, while a resolution higher than 1 Hz is desired, we have to divide a pulse into more units for counting. The present invention provides a solution to overcome the above problem by utilizing a phase lock loop (PLL) circuit. The PLL circuit generates a signal that has the same phase as the original one but has a counting frequency which is n times the original frequency. The relation between the new counting signal and the original one is illustrated in FIG. 6. The output signal (f) of the oscillation circuit of biosensor, is transmitted, via the PLL circuit, to a phase comparator for comparing the phase of signals. If the phase of the input signal (f) leads the signal at node (b), the signal at node (c) is a positive wave. The more the signal (f) leads the signal at node (b), the broader the pulse width of the signal at node (c) becomes. On tie contrary, as the input signal (f) phase lags behind the signal at node (b), the signal at node (c) is a negative wave. And, the less the signal (f) lags the signal at node (b), the narrower the pulse width of the signal at node (c) will be. When the phase difference between the signal (f) and the signal of node (b) is quite small, the signal at node (c) is a very narrow impulse After the signal at node (c) is filtered through a low pass filter, to generate a DC voltage signal to control the frequency of the output signal (nF) from voltage controlled oscillator (VCO). The higher the DC voltage input into the VCO, the higher the frequency of the output signal of the VCD (nf) becomes, and, after feedback, the frequency at node (b) will be raised. On the contrary, if the DC voltage is negative, the frequency at node (b) will descend. Eventually, the phase of the signal (nf) will be stabilized to be similar to the signal at node (b). Furthermore, the frequency of the output signal (nf) which is n times the frequency f of the signal f (namely, nf) is then transmitted to an ultra-high frequency counter, a high precision of The variation detected by the biosensor can be achieved. The operation principle of the combination of the phase lock loop (PLL) circuit with quartz crystal microbalance is further described below.

[0034] Essentially, the piezoelectric quartz crystal sensor utilizes oscillation to detect and identify the mass variation of target materials, so it intrinsically is all oscillator. The oscillation is converted into pulses. The number of the pulses are counted by a counter. In the example 1 described below, an oscillation with frequency (f) of 10 MHz is adopted for the purpose of illustration. About 10,000,000 pulses are counted in one second. However, the last one pulse, in fact, is not a complete pulse. Therefore, the actual count should be about 9,999,999 and ⅔ pulse:; And yet, the highest resolution is 1 A, because such a decimal fraction cannot be directly detected by directly inputting the oscillation with frequency (f) into the counters

[0035] The following examples are presented for illustration purposes and not to limit the scope of the invention.

EXAMPLE 1

[0036]

[0037] If we may incorporate and calculate the time of the last one pulse, φ, then we can raise the resolution of the bio-sensor. This invention employs the PLL circuit to achieve said purpose. The PLL circuit bas been used Lo produce a signal with the same phase as the original signal, but The frequency thereof has been raised n times in a cycle. As shown in Example 1, if the original frequency f equals “a+φ”, and “a” is an integer, since φ<1, the detectable frequency f is “a”. Now we raise the frequency to n times, nf, nf=na+b+φ′, wherein b=φ−φ′ and is an integer. So, if “na+b ” can be detected but φ′, which is less than 1, cannot be detected, (nf ) will be “na+b ”. If nF is divided by n, then we can get the original frequency f, (na+b)/n=a+(b/n). Therefore, we get the frequency count number of “a+(b/n)”. In other words, the resolution has been raised n times.

[0038] On the other hand, since the PLL circuit comprises a filter to trace the phase error and employs a close loop control to retain the phase relation ship. Even if the input signal is affected by noise to result in frequency jittering, the said PLL circuit will not produce any instantaneous change, and a stable efficacy can therefore be achieved. Such a result is an extraordinary and unexpected advantage of using PLL circuit.

[0039] According to FIG. 2, the signal frequency (f) of the oscillator is input to the PLL circuit, which generates an output with an n times frequency (nf). The signal nf is sent to an ultra-high frequency counter. The signal nf is sent Lo an ultra-high frequency counter. Subsequently, the ultra-high frequency counts the number of pulses produced in every second, thus a resolution of 1/n can be achieved. For instance, if n=100, the resolution will be 0.01. Since the present invention employs hardware to implement the high speed sampling, the variation sensed by of biosensor can be rapidly and precisely measured.

[0040] With respect to the block diagram and the waveforms shown in FIG. 4 and FIG. 5, one may compare the phases of the input signal (f) with the feedback signal at node (b) by a phase comparator. If the phase of signal (f) leads the phase of signal at node (b), the signal at of node (c) is a positive wave (the more the signal (f) leads, the broader the pulse becomes), If the phase of the signal (f) lags behind the phase of the signal at node (b), the signal at node (c) will be a negative wave (the less the signal (f) lags, the narrower the pulse becomes). When the phase differences between the signal (f) and the signal at (b) is quite small the signal at node(c) is a very narrow impulse. After the signal at node (c) is filtered through the low pass filter, a DC voltage is obtained at note (d). If the signal of (f) leads the signal at node (b) very much, the voltage at node (d) will be positive and very high. Such a high voltage is enlarged through a voltage amplifier to into an appropriate voltage (e) that is used to control and arise the output frequency (nf) of VCO. As a result, the frequency at node (b) is also raised. Eventually, when the phases of the signal (f) and the signal at node(b) become almost the same, a stable state is obtained. On the contrary, if the signal (f) lags behind the signal at node(b), the DC voltage at node (d) is negative and lowers the frequency the signal (f) until the phases of the signal (f) and the signal at node (b) become almost the same.

[0041] Because the division of frequency (nf) by n results in the frequency of the signal at node (b), which is then used in phase comparison, an output frequency (nf) which is n times the input frequency (f) can finally be obtained, and the phase difference between (f) and (nf) is a fixed value, Z, as shown in FIG. 6.

[0042] Furthermore, as illustrated in FIG. 4, the low pass filter may not only make the pulse width at node (c) correspond to the DC voltage at node (d), but also eliminate the noise. Therefore, the output of PLL possesses a merit of quite small jittering. Such a merit may overcome the problem of getting a worse S/N ratio after raising the resolution. This is all extraordinary advantage.

[0043] The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within The scope of the following claims. Without farther elaboration, it is believed that one skilled in the area can, using the preceding description, utilize the present invention to its fullest extent. Therefore, the examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows. 

What is claimed is:
 1. A high resolution biosensing system for detecting and identifying a biochemical material to be tested by using proportional relationship between frequency variation of oscillation and mass of the biochemical material to be tested, comprising: a biosensor for sensing mass of the biochemical material to be tested; an oscillator made by piezoelectric material and generating oscillation based on the sensed mass of the biochemical material to be tested; a phase-lock loop circuit for receiving the oscillation of the oscillator and generating pulse signals; an ultra-high frequency counter for counting the pulse signals from he phase-lock loop circuit; and a microprocessor and interface circuits for storing and controlling output from the ultra-high frequency counter, wherein said phase-lock loop circuit generates the pulse signals of a frequency, which is n times the frequency of the oscillator and with a constant phase difference there between, wherein n is a natural number greater than or equal to 1, to trigger the ultra-high frequency counter.
 2. The high resolution biosensing system as claimed in claim 1 , wherein the sensitivity of the biosensing system is above 10⁻¹⁰ g. 